Neuronal somatic plasmalemmal permeability and dendritic beading caused by head rotational traumatic brain injury in pigs-An exploratory study.

Closed-head traumatic brain injury (TBI) is induced by rapid motion of the head, resulting in diffuse strain fields throughout the brain. The injury mechanism(s), loading thresholds, and neuroanatomical distribution of affected cells remain poorly understood, especially in the gyrencephalic brain. We utilized a porcine model to explore the relationships between rapid head rotational acceleration-deceleration loading and immediate alterations in plasmalemmal permeability within cerebral cortex, sub-cortical white matter, and hippocampus. To assess plasmalemmal compromise, Lucifer yellow (LY), a small cell-impermeant dye, was delivered intraventricularly and diffused throughout the parenchyma prior to injury in animals euthanized at 15-min post-injury; other animals (not receiving LY) were survived to 8-h or 7-days. Plasmalemmal permeability preferentially occurred in neuronal somata and dendrites, but rarely in white matter axons. The burden of LY+ neurons increased based on head rotational kinematics, specifically maximum angular velocity, and was exacerbated by repeated TBI. In the cortex, LY+ cells were prominent in both the medial and lateral gyri. Neuronal membrane permeability was observed within the hippocampus and entorhinal cortex, including morphological changes such as beading in dendrites. These changes correlated with reduced fiber volleys and synaptic current alterations at later timepoints in the hippocampus. Further histological observations found decreased NeuN immunoreactivity, increased mitochondrial fission, and caspase pathway activation in both LY+ and LY- cells, suggesting the presence of multiple injury phenotypes. This exploratory study suggests relationships between plasmalemmal disruptions in neuronal somata and dendrites within cortical and hippocampal gray matter as a primary response in closed-head rotational TBI and sets the stage for future, traditional hypothesis-testing experiments.

Dietary manipulation of vulnerability to traumatic brain injury-induced neuronal plasma membrane permeability.

Traumatic brain injury (TBI) can produce physical disruptions in the plasma membranes of neurons, referred to as mechanoporation, which lead to increased cell permeability. We suspect that such trauma-induced membrane disruptions may be influenced by the physical properties of the plasma membrane, such as elasticity or rigidity. These membrane properties are influenced by lipid composition, which can be modulated via diet, leading to the intriguing possibility of prophylactically altering diet to confer resiliency to this mechanism of acute neuronal damage in TBI. In this proof-of-concept study, we used three different diets-one high in polyunsaturated fatty acids suggested to increase elasticity (Fish Oil), one high in saturated fatty acids and cholesterol suggested to increase rigidity (High Fat), and one standard rat chow (Control)-to alter brain plasma membrane lipid composition before subjecting rats to lateral fluid percussion injury (FPI). Lipid analysis (n = 12 rats) confirmed that diets altered brain fatty acid composition after 4 weeks of feeding, with the Fish Oil diet increasing unsaturated fatty acids, and interestingly, the High Fat diet increasing omega-6 docosapentaenoic acid. One cohort of animals (n = 34 rats) was assessed immediately after FPI or sham injury for acute changes in neuronal membrane permeability in the injury-adjacent cortex. Surprisingly, sham animals fed Fish Oil had increased membrane permeability, suggesting altered passive membrane properties. In contrast, injured animals fed the High Fat diet displayed less intense uptake of permeability marker, suggesting a reduced extent of injury-induced plasma membrane disruption, although the density of affected cells matched the other diet groups. In a separate cohort survived for 7 days after FPI (n = 48 rats), animals fed the High Fat diet exhibited a reduced lesion area. At both time points there were no statistically significant differences in inflammation. Unexpectedly, these results indicate that the High Fat diet, as opposed to the Fish Oil diet, beneficially modulated acute plasma membrane permeability and resulted in a smaller lesion size at 7 days post-injury. Additional studies are necessary to determine the impact of these various diets on behavioral outcomes post-TBI. Further investigation is also needed to understand the physical properties in neuronal plasma membranes that may underlie increased resiliency to trauma-induced disruptions and, importantly, to understand how these properties may be influenced by targeted dietary modifications for vulnerable populations.

Mechanosensation in traumatic brain injury.

Traumatic brain injury (TBI) is distinct from other neurological disorders because it is induced by a discrete event that applies extreme mechanical forces to the brain. This review describes how the brain senses, integrates, and responds to forces under both normal conditions and during injury. The response to forces is influenced by the unique mechanical properties of brain tissue, which differ by region, cell type, and sub-cellular structure. Elements such as the extracellular matrix, plasma membrane, transmembrane receptors, and cytoskeleton influence its properties. These same components also act as force-sensors, allowing neurons and glia to respond to their physical environment and maintain homeostasis. However, when applied forces become too large, as in TBI, these components may respond in an aberrant manner or structurally fail, resulting in unique pathological sequelae. This so-called "pathological mechanosensation" represents a spectrum of cellular responses, which vary depending on the overall biomechanical parameters of the injury and may be compounded by repetitive injuries. Such aberrant physical responses and/or damage to cells along with the resulting secondary injury cascades can ultimately lead to long-term cellular dysfunction and degeneration, often resulting in persistent deficits. Indeed, pathological mechanosensation not only directly initiates secondary injury cascades, but this post-physical damage environment provides the context in which these cascades unfold. Collectively, these points underscore the need to use experimental models that accurately replicate the biomechanics of TBI in humans. Understanding cellular responses in context with injury biomechanics may uncover therapeutic targets addressing various facets of trauma-specific sequelae.

Neurons in Subcortical Oculomotor Regions Are Vulnerable to Plasma Membrane Damage after Repetitive Diffuse Traumatic Brain Injury in Swine.

Oculomotor deficits, such as insufficiencies in accommodation, convergence, and saccades, are common following traumatic brain injury (TBI). Previous studies in patients with mild TBI attributed these deficits to insufficient activation of subcortical oculomotor nuclei, although the exact mechanism is unknown. A possible cause for neuronal dysfunction in these regions is biomechanically induced plasma membrane permeability. We used our established porcine model of head rotational TBI to investigate whether cell permeability changes occurred in subcortical oculomotor areas following single or repetitive TBI, with repetitive injuries separated by 15 min, 3 days, or 7 days. Swine were subjected to sham conditions or head rotational acceleration in the sagittal plane using a HYGE pneumatic actuator. Two hours prior to the final injury, the cell-impermeant dye Lucifer Yellow was injected into the ventricles to diffuse throughout the interstitial space to assess plasmalemmal permeability. Animals were sacrificed 15 min after the final injury for immunohistological analysis. Brain regions examined for cell membrane permeability included caudate, substantia nigra pars reticulata, superior colliculus, and cranial nerve oculomotor nuclei. We found that the distribution of permeabilized neurons varied depending on the number and spacing of injuries. Repetitive injuries separated by 15 min or 3 days resulted in the most permeability. Many permeabilized cells lost neuron-specific nuclear protein reactivity, although no neuronal loss occurred acutely after injury. Microglia contacted and appeared to begin phagocytosing permeabilized neurons in repetitively injured animals. These pathologies within oculomotor areas may mediate transient dysfunction and/or degeneration that may contribute to oculomotor deficits following diffuse TBI.

Installing Guest Molecules at Specific Sites within Scaffold Protein Crystals.

Protein crystals are porous self-assembling materials that can be rapidly evolved by mutagenesis. We aimed to develop scaffold assisted crystallography techniques in an engineered protein crystal with large pores (>13 nm). Guest molecules were installed via a single covalent bond to attempt to reduce the conformational freedom and achieve high-occupancy structures. We used four different conjugation strategies to attach guest molecules to three different cysteine sites within pre-existing protein crystals. In all but one case, the presence of the adduct was obvious in the electron density. Structure determination of larger guest molecules may be feasible due to the large pores of the engineered scaffold crystals.